Properties

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

Storage buffer

Preservative: 0.05% Sodium azide

Concentration information loading...

Purity

Ascites

Primary antibody notes

Voltage-sensitive calcium channels mediate the entry of calcium into many types of excitable cells and thus play a key role in neurotransmitter release and excitation-contraction (E-C) coupling. The 1,4-dihydropyridines (DHPs) are synthetic organic compounds which can be used to identify the L-type calcium channels that are found in all types of vertebrate muscle, neuronal and neuroendocrine cells. The DHP receptor is part of the L-type calcium channel complex and is thought to be the voltage sensor in E-C coupling.
The purified DHP receptor isolated from triads is composed of at least four subunits. The alpha-1 subunit contains the binding site for the DHPs and shows high sequence homology to the voltage gated sodium channel. The alpha-2 subunit is a large glycoprotein associated with the DHP receptor which was first described in skeletal muscle and is also found in high concentrations in other excitable tissues such as cardiac muscle and brain and in low concentrations in most other tissues studied. The other two subunits that co-purify with the DHP receptor are termed beta and gamma.

1/500. Detects a band of approximately 150 kDa (predicted molecular weight: 123 kDa).

Target

Function

The alpha-2/delta subunit of voltage-dependent calcium channels regulates calcium current density and activation/inactivation kinetics of the calcium channel. Plays an important role in excitation-contraction coupling.

Tissue specificity

Isoform 1 is expressed in skeletal muscle. Isoform 2 is expressed in the central nervous system. Isoform 2, isoform 4 and isoform 5 are expressed in neuroblastoma cells. Isoform 3, isoform 4 and isoform 5 are expressed in the aorta.

Images

Immunohistochemistry was performed on normal biopsies of deparaffinized human skeletal muscle tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer and microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a Mouse Monoclonal Antibody recognizing Calcium channel L type DHPR alpha 2 subunit (ab2864) or without primary antibody (negative control) overnight at 4�C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Flow cytometry analysis of Dihydropyridine Receptor alpha 2 showing positive staining in the membrane and cytoplasm of SH-SY5Y cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2864 at 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Flow cytometry analysis of Dihydropyridine Receptor alpha 2 showing positive staining in the membrane and cytoplasm of Neuro-2a cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a2864 at 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Flow cytometry analysis of Dihydropyridine Receptor alpha 2 showing positive staining in the membrane and cytoplasm of C6 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2864 at 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Immunohistochemistry was performed on normal biopsies of deparaffinized mouse skeletal muscle tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a Mouse Monoclonal Antibody recognizing Calcium channel L type DHPR alpha 2 subunit (ab2864) or without primary antibody (negative control) overnight at 4ºC in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

IHC-Fr image of anti-Calcium channel L type DHPR alpha 2 subunit staining with ab2864 on tissue sections from chicken hindbrain. The sections were blocked with 3% BSA for 1 hour at 4°C, before incubation with ab2864 (1/1000 dilution) for 16 hours at 4°C. The secondary was an Alexa-Fluor 488 conjugated goat anti-rabbit polyclonal, used at a 1/1000 dilution.

ab2864 at 1/250 staining mouse brain tissue sections by IHC (Formalin/PFA-fixed paraffin-embedded sections). The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed. The tissue was incubated with the antibody for 16 hours. A biotinylated goat polyclonal antibody was used as the secondary.

ab2864 at 1/500 staining rat spinal cord tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed, prior to incubation with the antibody for 16 hours. A biotinylated goat polyclonal antibody was used as the secondary.

This is a Mouse monoclonal [20A] to Calcium channel L type DHPR alpha 2 subunit so the host species of this primary antibody is mouse. The isotype is IgG2a so the compatible secondary antibody should recognize mouse IgG2a. The host species of the secondary antibody can be goat, rabbit, donkey, horse etc. If you wish to use enzyme-based detection system then the secondary antibody can be ALP or HRP-conjugated.

For further information, I would advise you to visit our Immunodetection site:

https://www.abcam.com/index.html?pageconfig=resource&rid=12853

If you need any further assistance in the future, please do not hesitate to contact me.

No epitope mapping has been completed for this product. The immunogen is rabbit dihydropyridine receptor. We do know that this product shows positive signal in Western Blot for mouse brain and IHC for rat brain. Could there be low expression in mouse N2a? Is it possible to retest with a higher ug amount on the Western Blot? I would be very happy to help work with you to achieve the best possible data using this product. If you could provide the details of your protocol including sample preparation, troubleshooting and any images, I will be very happy to review these for you.

This product is covered by our Abpromise guarantee. We are happy provide scientific support at any time. If you are using the product in species and applications listed on the datasheet and contact us within six months of purchase, we are also happy to replace or refund the product should we not be able to help you to resolve the issue. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Thank you for contacting us. Epitope mapping has not been performed for the monoclonal antibodies ab2864 and ab88486. The other antibodies are polyclonals and will bind at many different sites of the protein. Please let me know if I can be of further assistance.

The antibody, ab2864was made to a native protein, and therefore, it is not available as a synthetic blocking peptide. We unfortunately do not have any of the protein available to provide either. Please let me know if there is anything else I can help you with.

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this product.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
- Could you please specify the cell type of the mouse cells used for these experiments?
- Total cell lysates or membrane preparations have been run on the gel?
I look forward to hearing from you soon.

Thank you for your enquiry.
Ab2864 detects 1,4 dihydropyridine (DHP) receptor alpha 2 subunit from human, rat, mouse, guinea pig and rabbit skeletal and cardiac muscle.
To our knowledge, this antibody has yet to be tested in IHC on paraffin-embedded tissue sections. All tested applications are specified on Abcam product datasheets. If you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 Abcam Points will be offered for an image).
I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Please find the answers to each of your three questions below:
(1) We are not aware of this antibody having been used on paraformaldehyde fixed tissues. As you have no doubt seen from the datasheet, it has been used in IHC on frozen sections.
(2) I am unable to comment on the depth that the antibody will penetrate heart tissue as this will depend on the precise conditions of your experiment, nature of material used and how the tissue has been prepared
(3) Any anti IgG2a secondary will recognise this primary antibody. If you don't have a suitable antibody already in your lab, I'd certainly recommend that you try one of our recommended secondaries.